Astaxanthin-producing yeast cells, methods for their preparation and their use

ABSTRACT

An isolated pure culture of a strain of Phaffia rhodozyma which produces astaxanthin in an mount of at least 600  mu g per g Phaffia rhodozyma dry matter, as determined by HPLC analysis.

This is a divisional of application Ser. No. 07/919,986 filed Jul. 27,1992, now U.S. Pat. No. 5,356,810, which in turn is a continuation ofapplication Ser. No. 07/424,306 filed Dec. 11, 1989, now abandoned.

The present invention relates to astaxanthin-producing yeast strains,methods for their preparation, methods for their cultivation, andmethods for isolating the astaxanthin from the yeast cells. Further, theinvention relates to a food or feed which contains theastaxanthin-containing yeast cells or astaxanthin recovered from theseas well as a method for producing food or feed and a method for feedinganimals with said feed.

It is known that the red colour of the meat of anadromous fish such assalmon or sea trout is due to red pigments such as astaxanthin which ispresent in the feed consumed by the fish. In natural surroundings, thefish obtain their red colour from crustaceans or otherastaxanthin-containing organisms, but when being bred in fish farms, thefish do not have access to these natural pigmentation sources andtherefore do not obtain the attractive red colour unless red pigmentsare supplied in the feed.

Thus, astaxanthin isolated from crustacean wastes or producedsynthetically as well as other synthetic red pigments such ascantaxanthin have been used as constituents in fish feed. However, theuse of cantaxanthin in animal feedstuffs is prohibited in certaincountries, and the synthetic astaxanthin production as well as theprocess for isolating natural astaxanthin are rather expensive and oftenalso subject to seasonal variations.

Other natural astaxanthin sources are known, among these the yeastPhaffia rhodozyma and some microalgae such as the unicellular group ofgreen algae Chlamydomonas nivalis Gert Knutson et al., "Pigmentering aflaks med astaxanthin fra mikroalger", Norsk Fiskeopdrat nr. 3, pp. 4-6,55 (1980)!. The astaxanthin produced by these organisms has been shownto confer the desired red colour to anadromous fish Eric A. Johnson etal., "Phaffia rhodozyma as an astaxanthin source in salmonid diets",Aquaculture, 20, pp. 123-134 (1980) and JP-A 57-206342!. However, theuse of yeast cells in large amounts as nutrition for the fish is notdesirable as this feed is not sufficiently varied. On the other hand,the amount of astaxanthin produced by the organisms and present in anutritionally acceptable amount of yeast cells is not sufficient toobtain the desired pigmentation, and the isolation of astaxanthin fromyeast by the known methods is rather expensive.

If, however, it would be possible to obtain a higher astaxanthinproduction from these organisms, a profitable astaxanthin productionwhich is not subject to seasonal conditions would be possible.

The present invention provides yeast cells which contain astaxanthin insufficiently high concentrations to make it possible to use the yeastcells as or in feed for anadromous fish and other animals in which apigmentation of the animal meat or a product of the animal is desired.The invention also provides attractive methods for obtaining astaxanthinfrom astaxanthin-containing yeast cells, in particular theabove-mentioned yeast cells having high contents of astaxanthin.Important aspects of the invention are based on a particular method forcultivating astaxanthin-producing cells and/or the provision of mutantstrains with an improved inherent capability of producing astaxanthin.

Thus, one aspect of the invention relates to a yeast cell which, whengroom under conditions comprising an oxygen transfer rate of at least 30mmoles/1/hour on Difco YM medium at 20°-22° C. for 5 days in 500 mlshake flasks with two baffles containing 50 ml of the medium andsubjected to orbital shaking at 150 rpm, the inoculum being 100 μl of afour days old culture in YM-medium, produces astaxanthin in an amount ofat least 300 μg per g of yeast dry matter, determined by HPLC analysisusing pure astaxanthin as a standard on a methanol extract of the yeastprepared by subjecting a suspension of 0.2 g of yeast dry matter in 20ml of methanol to 5×1 minutes of disintegration at intervals of half aminute, the disintegration being performed at a temperature of at themost 20° C. in a glass ball mill containing 15 g of glass balls having adiameter of 0.4 mm, the glass ball mill being provided with a coolingjacket with ice water.

The growth conditions and determination conditions stated above aregiven to standardize the growth and testing methods so that the resultobtained will reflect the inherent astaxanthin-producing capabilities ofthe yeast in question. This method has been found, by severalexperiments performed by the applicant company, to be a suitable andreproducible method which is easy to perform in practice. It should benoted that the determination method is not the same as the one hithertoused in the literature. The methods hitherto used in the literature, cf.e.g. Eric A. Johnson et al., "Astaxanthin formation by the yeast Phaffiarhodozyma", Journal of general microbiology 115, 1979, pp. 173-83, arebased on the absorbance of a 1% (w/v) solution in acetone in a 1 cmcuvette of 1600, whereas the value, which is obtained by measuring theastaxanthin standard from Hoffmann-La Roche, is 2100. This value isbased on the applicant company's own measurements as well as on theinformation given by Hoffmann-La Roche.

Furthermore, the known determination method measures the total pigmentcontent of the yeast whereas the above-mentioned standard method used inthe present application exclusively measures the astaxanthin content.When comparing the values obtained by the standardized method as statedabove with the values stated in the literature, it should be borne inmind that the values stated in the literature will be considerablyhigher than the true values obtained by the standardized method statedabove. Thus, whenever the present total pigment content is compared withthe literature statements, a direction for the difference in extinctioncoefficients should be made by multiplying the total pigment contentstated in the literature by 1600/2100.

The growth conditions stated above are the ones which have been found bythe applicant company to be reproducible and significant for thedetermination of the inherent astaxanthin-producing capability. A moredetailed explanation of the growth and determination conditions used fordetermining the inherent astaxanthin-producing capabilities of yeaststrains is given in connection with the Examples.

The yeast cell according to the invention is preferably a yeast cellwhich belongs to the genus Phaffia and in particular one which belongsto the species Phaffia rhodozyma as this is the only Phaffia speciesknown for the time being.

At present, Phaffia rhodozyma is the only known yeast which producesastaxanthin. The wild-type P. rhodozyma is isolated from deciduous treeexudates and an example of such a wild-type strain is deposited in theAmerican Type Culture Collection under the accession number ATCC 24261.

Vegetative P. rhodozyma cells form buds as heterobasidiomycetous yeast.Clamydospores are developed by budding but promycelium and proper sporeformation do not occur. The chlamydospores are relatively largespherical cells with a larger lipid content than the vegetative cells.Attempts to pair the various strains in the hope of observing dikaryoticmycelium and teliospore formation have not been successful. P. rhodozymawas therefore classified in the genus Deuteromycotina of the orderBlastomycetes (cf. M. W. Miller et al., "Phaffia, a new yeast genus inthe deuteromycotina (Blastomycetes)", in International Journal ofsystematic bacteriology 26:2, 1976, pp. 286-291).

Vegetative cells are ellipsoidal (3.6-7.5)×(5.5-10.5) μm and are presentin a liquid medium individually, in pairs and in some cases in shortchains or small clusters. No true mycelium is developed, but arudimentary pseudomycelium may be present. Budding occurs several timesfrom the same point on the cell. P. rhodozyma has a strong cell membranecomposed by many layers, and capsule material imparts a granularappearance to the surface and causes the clustering mentioned above.

A sexual cycle of life has not been observed. During the development ofthe chlamydospores, vegetative cells are formed by budding. These cellscannot be considered to be promycelia with spores as described forAessosporon (cf. J. P. van der Walt, "The perfect and imperfect statesof Sporobolomyces salminicolor", J. Microbiol. Serol. 36, 1970, pp.49-55). The chlamydospores cannot be considered to be gonotoconter(sexually segregated spores), and their buds cannot be considered to bethe haploid generation. It has not been possible by nuclear staining todemonstrate diploidization at any growth stage. Transmission electronmicrographs have only shown one single nucleus during all growth phases(cf. M. W. Miller et al., op.cit.). Thus, P. rhodozyma is likely to behaploid, but this has not been proved.

After 2-4 weeks of growth on YM agar (Difco Laboratories Incorporated,Difco manual: dehydrated culture media and reagents for microbiology,10th Edition, Detroit 1984), the string cultures are orange tosalmon-pink, depending on the strain.

P. rhodozyma has the special property of not growing at temperaturesabove 27° C. It ferments D-glucose, maltose, sucrose and raffinosewhereas D-galactose and melibiose are not fermented. The most commoncarbon sources are assimilated: however, D-galactose, L-sorbose,melibiose, lactose, glycerol and citrate are not assimilated. The yeastcannot grow in vitamin-free medium without the addition of biotin (M. W.Miller et al., op.cit.). The most common nitrogen sources areassimilated, including urea. Potassium nitrate and ethylamine are notassimilated. The yeast cannot grow on 50% by weight of a glucose-yeastextract agar nor on 10% by weight of sodium chloride-yeast extract agar.The acid formation on chalk agar by the yeast is weak and so is thegelatin liquefaction. Casein hydrolysis, depolytic activity and growthin the presence of 0.1 μg of cycloheximid per ml are absent whereas theyeast is able to synthesize starch-like compounds independent of pH. Themole-% G+C is measured to be 48.3±0.18 (cf. Miller et al., op. cit).

During growth under carbohydrate- and/or nitrogen-limited conditions,when subjected to fed-batch fermentations, P. rhodozyma producestrehalose as a carbohydrate deposit. This is quite a new observationmade by the applicant company and has not been reported hitherto.

P. rhodozyma produces a number of carotenoids, of which astaxanthinconstitutes 83-87%, β-carotene 2-2.5%, echinenone 2-4% andphoenicoxanthin 5-7%, according to the literature. In practice, theratio of astaxanthin to total pigment produced by P. rhodozyma has,however, been found to vary considerably depending on the growthconditions of the yeast cells as well as the pigment determinationmethod and has generally been found to be in the range of 50-80%.##STR1## All hydroxylated pigments, including astaxanthin, have beendescribed as non-bound, not as esters or other derivatives (Arthur G.Andrewes et al., "Carotenoids of Phaffia rhodozyma, a red-pigmentedfermenting yeast", in Phytochemistry 15, 1976, pp. 1003-1007). Thereexist three optical isomeric forms of astaxanthin: (3S,3'S), (3R,3'R)and (3S,3R), each existing in various trans- and cis-configurations. Ithas been reported that P. rhodozyma only produces (3R,3'R)-astaxanthin(Arthur G. Andrewes et al., "(3R,3'R)-astaxanthin from the yeast Phaffiarhodozyma", op. cit., pp. 1009-1011). In the present context,"astaxanthin" is used about trans- as well as cis-configurations ofastaxanthin.

The pigment in the individual P. rhodozyma cells is not visible when thecells are studied in a microscope, which indicates that the pigment maybe dispersed throughout the cell. However, it is also possible that thepigment is concentrated in certain parts of the cells.

Astaxanthin is an oxidated carotenoid and therefore belongs to thexanthophyl group. Similarly to other carotenoids, astaxanthin iscomposed of eight isoprenoid units. By the biosynthesis of astaxanthinwhich is catabolite repressed, isopentenyl pyrophosphate is formed fromacetyl-CoA as illustrated below. ##STR2##

By three prenyltransferase reactions, isopentenyl pyrophosphate formsgeranyl geranyl pyrophosphate via geranyl pyrophosphate and farnesylpyrophosphate as illustrated below. ##STR3##

Condensation of two-molecular geranyl geranyl pyrophosphate formsphytoene which, via dehydrogenation steps and ring forming, formsastaxanthin from H-carotene. The last part of the biosynthesis has notbeen unambiguously determined, but Andrewes et al. (op.cit.) haveproposed the metabolism route shown below on the basis of the pigmentcomposition in P. rhodozyma. ##STR4##

The enzyme system which converts geranyl geranyl pyrophosphate toastaxanthin is not known, and it is therefore not known why P.rhodozymaproduces (3R,3'R)-astaxanthin and whether there are someregulating steps during this part of the biosynthesis. On the otherhand, the conversion of acetyl-CoA to isopentenyl pyrophosphate in otherisoprenoid-producing organisms than P. rhodozyma and the enzymes whichtake part have been described in relatively great detail. Less is knownabout the enzymes which convert isopentenyl pyrophosphate to geranylgeranyl pyrophosphate (isopentenyl pyrophosphate isomerase andprenyltransferase (J. W. Porter, S. L. Spurgeon (eds.). "Biosynthesis ofisoprenoid compounds". New York, 1981-1983).

The protein content of P. rhodozyma varies from 25 to 50% of yeast drymatter, depending on the culturing conditions. This is a relatively lowprotein content. In contrast to this, the lipid content isextraordinarily high (14-27%). It is contemplated that the nucleic acidconstitutes 8% similarly to other yeasts and that the amino acidcomposition is similar to the composition in other known yeasts such asSaccharomyces cerevisiae and thus has a very low content of certainamino acids, e.g. methionin and cystein (cf. Gerald Reed and Henry J.Peppler, Yeast Technology, 1973, p. 329, published by The AVI PublishingCompany, Inc.). This and the overall yeast composition which comprises ahigh amount of nucleic acids make the yeast inconvenient for animalnutrition purposes when the yeast is the only nutrient source, such asindicated above. Thus, without addition of certain amino acids and othernutrient components, the yeast will not be a suitable major nutritioncomponent for fish or other animals.

The total amount of astaxanthin which is produced by the wild type P.rhodozyma when this is grown under the normal known conditions issufficient to confer a red colour to the yeast cell but is notsufficient to make recovery of the astaxanthin from the yeast cellseconomically feasible.

None of the Phaffia rhodozyma species described in the literature havean inherent astaxanthin-producing capability of more than 300 μg per gof yeast dry matter when analyzed in accordance with the above standardmethods, vide Table 2 and Table 6 of the examples. However, according tothe present invention, it has been found possible to obtain yeast cellswhich are inherently capable of producing astaxanthin in an amount of atleast 450 μg per g of yeast dry matter, such as at least 600 μg per g ofyeast dry matter, preferably at least 700 μg per g of yeast dry matter,more preferably at least 1000 μg per g of yeast dry matter, especiallyat least 1500 μg per g of yeast dry matter, and most preferably at least2000 μg per g of yeast dry matter, the growth and the determinationbeing performed by the standard methods stated above. These yeast cellshave been produced from naturally occurring Phaffia rhodozyma bymutagenization. Thus, an aspect of the present invention relates to amethod for producing a yeast cell showing the high inherentastaxanthin-producing capability explained above, the method comprisingtreating a yeast cell with a mutagen and selecting a resulting mutantwhich, when grown under the conditions stated above, is capable ofproducing astaxanthin in an amount of at least 300 μg per g of yeast drymatter, determined by the method stated above.

The mutagenization may be performed as a single mutagenization, but ithas been found advantageous to perform two or more consecutivemutagenizations, as it has been found that the inherent capability ofproducing astaxanthin may be improved by each mutagenization step. Thestarting yeast cell subjected to mutagenization is normally a yeast cellwhich, when grown under the conditions stated above, producesastaxanthin in an amount of less than 300 μg per g of yeast dry matter,determined by the method stated above, but it is evident that a normalcandidate for the mutagenization treatment will be a naturally occurringyeast cell having as high inherent astaxanthin production as possible.Such yeast cells are normally yeast cells which belong to the genusPhaffia, in particular yeast cells belonging to the species Phaffiarhodozyma, such as is mentioned above.

The mutagenization treatment may be performed using any suitable mutagen(in the present context, the term "mutagen" is to be understood in itsbroad sense as comprising. e.g., not only agents which have a mutageneffect, but also treatment which have a mutagen effect such as UVirradiation). Examples of suitable mutagens are ethyl methanesulphonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine,nucleotide base analogues such as bromouracil, and acridines, but it iscontemplated that any other effective mutagen will be suitable for thetreatment.

In accordance with conventional mutagenization techniques, themutagenization is followed by suitable selection of the cells which havethe highest astaxanthin production. Due to the fact that astaxanthin isa pigment, this selection may be performed relatively easily by normalvisual means, such as simple observation of single colonies. Analternative method is to perform analysis on cultures made from singlecolonies, e.g. by using the standardized cultivation conditions anddetermination conditions as explained above.

Two strains produced by the mutagenization method according to theinvention and showing a particularly high astaxanthin productivity weredeposited on 6 Apr., 1987 at the Centraalbureau voor Schimmelcultures,Oosterstraat 1, Postbus 273, NL-3740 AG Beam, the Netherlands (CBS)under the accession Nos. 224-87 and 225-87, respectively, and one strainbeing a reisolate of CBS 225-87 (vide Example 1) was deposited on 23Mar., 1988 an CBS under the accession No. 215-88, and an aspect of theinvention relates to these yeast strains as well as mutants orderivatives thereof which have substantially retained or improved theastaxanthin-producing capability of these strains.

The invention also relates to a method for producingastaxanthin-containing yeast cells or cell parts, or astaxanthin derivedfrom these yeast cell or cell parts. This method comprises cultivatingastaxanthin-producing yeast cells under aerobic conditions in a mediumcontaining carbohydrate sources, assimilable sources of nitrogen andphosphorus, micronutrients and biotin or desthiobiotin at a temperaturein the range of 15°-26° C. so as to obtain a biomass containingastaxanthin in an amount of at least 300 μg per g of yeast dry matter,determined by the method stated above, and optionally performing one orseveral of the following steps in arbitrary sequence:

harvesting cells from the culture so as to obtain a yeast cream, openingthe cells, for example rupturing the cell walls by means of mechanical,chemical and/or enzymetic treatment and/or subjecting the cells tosonication, autolysis, osmolysis and/or plasmolysis optionally withaddition of suitable agents such as detergents, acids, bases, enzymes,autolysis-enhancing substances, osmolysing agents such as salts, and/orplasmolysing agents,

homogenizing the cells to obtain a homogenate,

drying the cells, the cell fragments or the homogenate, preferably to awater content of at the most 12% by weighs, preferably at the most 10%by weight,

extracting astaxanthin from the cells, the cell fragments or thehomogenate.

The amount of astaxanthin stated above, 300 μg per g of yeast drymatter, is higher than any astaxanthin concentration reported in theliterature. Although Johnson et al., op. cit., reports an astaxanthincontent of 295 μg per g of yeast dry matter, this value does not onlycomprise the astaxanthin content but in fact the total pigment contentof the yeast cell. Further, this pigment content was measured using avalue of the absorbance of a 1% (w/v) solution in acetone in 1 cmcuvette of 1600 which is lower than the one measured by the presentapplicants (2100) so that the value reported by Johnson et al.corresponds to at the very most 295×1600/2100-225 μg of total pigment(not only astaxanthin) per g of yeast dry matter. This pigment contentfrom the literature should be compared with the total pigment content ofthe yeast strains of the present invention which is 885 μg/g of yeastdry matter for the strain CBS 224-87, 1176 μg/g of yeast dry matter forthe strain CBS 225-87, and about 1340 μg/g of yeast dry matter for thestrain CBS 215-88, or even higher, e.g. at least 2000 μg/g of yeast drymatter.

The high astaxanthin concentration in the yeast cells of the inventionmay be obtained partly by the use of special cultivation conditions asexplained below and partly by selecting a yeast strain with a highinherent astaxanthin productivity, preferably a yeast strain asdiscussed above, and in particular it is preferred to combine thespecial cultivation conditions and the use of specialastaxanthin-producing yeast strains.

The cultivation is preferably performed as a fed-batch fermentationunder conditions where substantially no alcohol is formed. As mentionedabove, the temperature of the culture is in the range of 15°-26° C.Below 15° C., the growth tends to be too slow to be acceptable forindustrial production, and above 26° C., the viability of the culture isseverely impaired. The preferred temperature range is 20°-22° C.

The fermentation or at least part thereof is normally performed in amedium which comprises suitable macro- and micronutrients for the cells,such as molasses or saccharose as a carbohydrate source and nitrogensources such as corn-steep-liquor, diammonium sulphate, Ammoniumphosphate, ammonium hydroxide or urea, phosphorus sources such asammonium phosphate and phosphoric acid and added micronutrients ormineral salts such as magnesium sulphate, zinc sulphate and biotin ordesthiobiotin. The molasses or saccharose are preferably supplied to themedium separately from the other components in accordance withconventional methods used in yeast production. When the medium comprisesmolasses, it has been found that the growth of the yeast cells isaffected by the concentration of sugar or other growth-inhibitingsubstances therein in the fermenter. This effect has not been observedwhen the medium comprises corn-steep-liquor or solids. Accordingly, itmay prove advantageous to regulate the fermentation so that theconcentration of sugar (expressed as the total concentration of glucoseand saccharose) in the fermenter is at the most 8 g/l, preferably at themost 5 g/l, and most preferably at the most 1 g/l.

The culture is aerated during the total fermentation, i.e. it is grownunder aerobic conditions. By the term "aerobic conditions" is meant thatthe oxygen supply should be sufficient so that substantially no oxygenlimitation will occur during the fermentation.

According to a special aspect of the invention as indicated above, theconcentration of astaxanthin in the biomass obtained is increased byperforming the cultivation under selected conditions. These conditionsinvolve a cultivation which comprises a growth phase under conditionswhich are substantially sufficient with respect to substantially allgrowth conditions and a subsequent growth-limited phase. Thegrowth-limited phase is preferably established by providing conditionswhere the growth medium under continued aeration is deprived of at leastone growth factor so as to enhance the production of taxanthin duringthe subsequent phase.

The growth-limited phase should be understood to generally mean thephase in which the main part of the cells have stopped growing. Thisphase does of course occur when the medium is deprived of at least onegrowth factor but is also observed during the last part of the period ofcarbohydrate addition when the amount of cells present in the fermenteris well in excess of the aeration capacity of the fermenter.

It is not known why the subsequent growth-limited phase has thesurprising effect of considerably enhancing the production ofastaxanthin (for example from 231 to 369 μg per g of yeast dry matter asobtained in one of the examples which follows), but it is contemplatedthat the precursors of astaxanthin have been produced during the growthphase, and that the subsequent growth-limited phase provides conditionswhich promote the final production of astaxanthin, possibly oxidizingconditions with the surplus of oxygen which becomes available when thegrowth is terminated. At any rate, it seems essential that aeration iscontinued during the subsequent growth-limited phase. The duration ofthe subsequent growth-limited phase is preferably at least about 16hours, such as 16-24 hours, as shorter durations may tend to decreasethe extra effect obtainable, whereas there seems to be no substantialeffect obtainable by extending the growth-limited phase to more thanabout 24 hours.

Expressed in a functional manner, the conditions of the growth-limitedphase should be adapted to enhance the astaxanthin production to atleast 1.2 times the production obtained without the subsequent phase,such as at least 1.3 times the production obtained without thesubsequent phase, preferably at least 1.4 times the production obtainedwithout the subsequent phase and most preferably at least 1.5 times theproduction obtained without the subsequent phase.

While the yeast cell subjected to the special cultivation with thesubsequent growth-limited phase may be a wild-type astaxanthin-producingyeast cell whose astaxanthin productivity is increased due to thesubsequent growth-limiting step, such as a wild-type yeast cell of thegenus Phaffia, in particular of the species Phaffia rhodozyma, it ispreferred that the yeast cell subjected to the cultivation is a yeastcell having an inherent and improved capability of producingastaxanthin, typically a yeast cell obtained by mutagenization asexplained above. With these yeast cells with an inherent increasedastaxanthin production, the concentration of astaxanthin in the biomassobtained when using the special cultivation method comprising agrowth-limited phase may be at least 600, preferably at least 800, morepreferably at least 1000 μg per g of yeast dry matter, especially atleast 1500 μg per g of yeast dry matter, e.g. at least 2000 μg per g ofyeast dry matter, and most preferably at least 3000 μg per g of yeastdry matter, determined as stated above.

Normally, and as used above, the total pigment content and astaxanthincontent of the yeast cells or yeast cell parts are stated as μg/g ofyeast dry matter. However, other ways of stating the total pigment andastaxanthin content may be found convenient. It may, e.g., be useful tostate the total pigment content and astaxanthin content as μg/ml of thesuspension in which it is present, e.g. in the growth media. Thereby, itwill not be necessary to determine the weight of yeast dry matter of theyeast cells from which the astaxanthin or total pigment is recovered.Thus, during the fermentation or cultivation of the yeast cells, theastaxanthin and/or total pigment content of the yeast cells may easilybe determined.

After the cultivation as described above to obtain yeast cells having ahigh astaxanthin content, the culture may be subjected to the subsequenttreatments mentioned above to isolate the yeast cells and/or conditionthem for their subsequent use, such as by rupturing the cells, and/orastaxanthin may be extracted from the cells.

The following important examples of these treatments are discussed ingreater detail:

The cells may be ruptured by subjecting the cells to an increasedpressure and then releasing the pressure.

The cells may be subjected to the increased pressure and release of thepressure by passage through a system comprising a valve homogenizerwhere the increased pressure is built up in front of the valvehomogenizer. The valve homogenizer typically comprises an aerojetthrough which the cell suspension is passed under high pressure and anobstruction member which the jet hits substantially after passagethrough the valve. Examples of cell disruption valves are described inAPV Gaulin Technical Bulletin No. 74 of March 1985 (APV GaulinInternational SA. P.O. Box 58, 1200 AB Hilversum, the Netherlands),incorporated by reference herein. As an example of a suitable cellrupture homogenizer may be mentioned an APV Gaulin MC4 homogenizer witha cell rupture valve of the type CR as described in the above-mentionedpublication. The homogenizer is connected to a a heat exchanger in whichthe suspension comprising ruptured cells passes from the cell rupturevalve. The pressure of the cell suspension in front of the valve may,e.g., be about 400-1200 bar. such as, e.g., about 700 bar. Thistreatment may for example be repeated three times with interveningcooling of the homogenate in the heat exchanger.

As is explained below, it is necessary that the cells are ruptured orotherwise treated when they are to be used in feed as the utilization ofthe astaxanthin content to a high degree depends on the cell contentsbeing available to the digestive system of the animal in question. Thus,substantially no pigmenting effect is obtained when feeding fish withfeed containing non-ruptured astaxanthin-containing cells.

The ruptured yeast cells may be subjected to ultrafiltration orevaporation so as to concentrate the ruptured cells. The ultrafiltrationmay, e.g., be performed in a lab unit system available from De DanskeSukkerfabrikker, for example a System 37 which comprises three filteringunits of a total filter area of 0.88 m² of an ultrafiltration membraneof the type RC 70. Another method for concentrating the ruptured cellsis to perform vacuum evaporation of water from the cell suspension.

The ruptured cells may be dried by spray drying or drum drying. Beforedrying, carriers such as sodium caseinate, antioxidants and/oremulsifiers are preferably added. Spray drying may, e.g., be performedby subjecting a homogeneously mixed slurry of the ruptured cells andoptionally a carrier such as sodium caseinate, preferably in the form ofan aqueous solution, to spray drying. The spray drying may suitably becarried out by mixing the aqueous sodium caseinate solution with theyeast slurry so as to obtain a sodium caseinate concentration of about2-10% (w/v). The resulting mixture is then allowed to stand withstirring in a nitrogen atmosphere before being pumped into a spraydrying tower in which it is subjected to drying at a temperature of,e.g., 150°-230° C., such as about 180° C. to decrease the water contentof the yeast cell material to e.g. at the most 10% by weight. The yeastcell material is then subsequently atomized by means of a spray wheel.The powdery yeast material resulting from the spray drying treatment issuitably recovered by means of cyclone and optionally subsequentlysieved and packed. An example of a suitable spray drying equipment is aspray tower of the type EAK-1 from Anhydro. As an alternative, theruptured yeast cells may be subjected to drum drying, for example in aclosed drum drying equipment at a temperature of 150°-200° C.

As the astaxanthin is very easily decomposed at high temperatures, it isimportant that the ruptured yeast cells are subjected to hightemperatures for as short a time as possible. Further, as astaxanthin issensitive to oxygen, the drying should preferably be performed undernon-oxidizing conditions, for example in an inert atmosphere such aswater vapour (which may be the water vapour evaporated from the yeastsuspension), nitrogen, and/or carbon dioxide.

Prior to drying, the ruptured cells are optionally mixed with suitableemulsifiers such as sorbitan monostearate or antioxidants, butylhydroxytoluene (BHT), butyl hydroxyanisol (BHA), vitamin E, ascorbicacid, (II) sulphate or (II) phosphate esters of ascorbic acid, orascorbyl palmitate.

Dried ruptured cells are immediately useful as a constituent of animalfeed, such as is explained below.

The astaxanthin content of the yeast cell may be extracted from these byuse of various extraction agents and extraction procedures--so as toensure that a substantial total extraction of the astaxanthin from theyeast cells is obtained. In most cases, the extraction has to beperformed in ruptured cell material. Thus, the ruptured cell material,which may be dry or wet, may be extracted with an organic solvent suchas petroleum ether which is suitably employed in the case of wet cellmaterial as the petroleum ether forms a phase separately from the waterphase. Other suitable organic solvents are acetone or alcohols such asmethanol or ethanol, ethers, ketones and chlorinated hydrocarbons. Bythe extraction, astaxanthin is dissolved in the organic solvent. Theastaxanthin may be obtained by removing the solvent from the solutionsuch as by evaporation in a falling film evaporation system beforedrying. However, also a concentrate of astaxanthin in the organicsolvent may be convenient for certain purposes. A concentrate may beused per se in the production of feed or food, or the concentrate may bediluted and used in the diluted state in the preparation of feed orfood, for example by impregnating feed or food constituents with thesolution or by using the solution (or the concentrate) for colouringfood constituents such as oils or fats.

The astaxanthin may also be extracted from yeast cells by use ofcarbondioxides under supercritical conditions. The carbondioxide mayoptionally be used in combination with suitable entrainers such asorganic solvents, especially solvents of the above mentioned types, orsolvents such as chloroform or acetonitrile, or glacial acetic acid. Theyeast cells subjected to supercritical extraction may be wet or drywhole yeast cells or ruptured, e.g. homogenized, yeast cells.

A preferred method of isolating whole astaxanthin-containing cells fromthe culture is to filtrate the yeast cream, for example on a filterpress or a rotating drum filter, so as to obtain a filter cake, e.g.with a dry matter content of about 25-35%. The filter cake may thensuitably be extruded into strings, for example strings with a diameterof about 0.5-2.0 mm in an extruder equipped with a perforated plate, soas to obtain strings consisting of yeast particles. The strings arepreferably extruded directly into the hot air in a fluid bed where theyare dried. The evaporation in the fluid bed is preferably regulated sothat the temperature of the yeast particles is kept below 50° C. such asat 30°-40° C., and the process is terminated when the water content isbrought down below 10% by weight, preferably below 8%, as determined bythe yeast dry matter content (the procedure is described in theExamples). Alternatively, the drying may be performed in a tray drierunder the same conditions as in the fluid bed. The dried whole cellmaterial may then be comminuted in a ball stirring mill such as aCoball® mill after which it is subjected to extraction.

According to a special method, whole cell dried material, for exampleobtained as described above, may be mixed with an oily phase such as anedible oil or fat such as soy bean oil or fish oil, or another organicsolvent such as a solvent of the type discussed above. The temperatureis preferably in the range of 20°-30° C. The mixture obtained from thecell material and oily phase or the organic solvent may be ground in amill such as a ball mill, e.g. a ball stirring mill such as a Coball®mill, to rupture the cells and release astaxanthin from the cells. Theresulting suspension may be used as such in feed, or the oily phasecontaining the astaxanthin may be separated from cell residues beforeuse. The separation is suitably performed by centrifugation in a fastrunning centrifuge, the same principle which is employed in separationof bacteria from wort. Another possibility is of course to mix ruptureddried cell material obtained by the methods discussed above with an oilyphase in a similar manner to extract the astaxanthin into the oily phaseand perform separation as described above. The oily phase may be usedfor colouring feed in the same manner as described above.

In contrast to most conventional extraction procedures which, as statedabove, has to be performed on ruptured cell material, it has been foundthat glacial acetic acid successfully may be employed to extractastaxanthin from whole, non-ruptured yeast cells. Thus, according to oneaspect of the present invention, astaxanthin may be extracted from wholeyeast cells with a solvent comprising glacial acetic acid, theextraction preferably being performed at a temperature above thefreezing point of the solvent, e.g. in the range of 20°14 100° C.,preferably in the range of 20°-80° C., and more preferably in the rangeof 20°-60° C. It is contemplated that it is possible to obtain a moreselective extraction of astaxanthin when the extraction is performed atthe lower temperatures as concomitant extraction of fat and otherextractable components will be limited at these low temperatures. Theconcentration of glacial acetic acid in the solvent is preferably in therange of 5-100, 10-70. the extraction with glacial acetic acid resultsin an extraction of the pigment of the cells of about 70-90%, i.e.substantially all the pigment and astaxanthin contents of the yeastcells are found in the glacial acetic acid extract. In addition, theextract normally contains about 30-35% of yeast dry matter. Suitably,the yeast subjected to extraction with glacial acetic acid is in theform of dried yeast, e.g. yeast which has been filtered and subsequentlyextruded into a fluid bed wherein it is dried, as thus treated yeastcells will not rupture during the extraction treatment (unless theextraction treatment involves vigorous mechanical treatment of the yeastcells). This will facilitate the subsequent separation of the extractcontaining the pigment from the yeast cells as compared with extractionof ruptured or homogenized cells, which, due to their relatively smallsizes in comparison with non-ruptured cells to a large extend tend toblock up the pores of the filter employed. The glacial acetic acidextraction is illustrated in Example 9. Extraction of wet yeast cellswith glacial acetic acid may also prove useful.

The extracted astaxanthin as well as the whole dried cell material arepreferably kept under oxygen-deficient conditions so as to protect theastaxanthin from decomposition. Thus, the astaxanthin-containing yeastcells or the extracted astaxanthin is preferably protected by means ofantioxidants such as butyl hydroxyanisol (BHA), butyl hydroxytoluene(BHT), vitamin E or ascorbic acid. (II) sulphate or (II) phosphateesters of ascorbic acid, or ascorbyl palmitate, and/or emulsifiers suchas monoglycerides or sorbitan esters and are suitably kept underhermetic conditions.

The invention also relates to an animal feed comprising yeast cells oryeast cell parts containing astaxanthin in an amount of at least 300 μgper g of yeast dry matter, determined as explained above in combinationwith other feed constituents. Preferably, the astaxanthin-containingyeast cells or yeast cell parts constitute at the most 10% by weight ofthe dry matter of the total animal feed composition, preferably at themost 5% and more preferably at the most 3%. These values are calculatedon the final feed to be administered to the animals. It is also possibleto prepare feed premixes having a higher concentration of yeast cells.The yeast cells or yeast cell parts or the astaxanthin is optionallyadmixed with emulsifiers which are capable of making the astaxanthindispersible in water. In addition, the astaxanthin-containing yeastcells or yeast cell parts may be protected against oxidation by means ofthe antioxidants and/or emulsifiers mentioned above, and/or the animalfeed may be packaged in air-tight and optionally evacuated containers.

The astaxanthin-containing dried yeast cells may also be packaged per sefor use as a feed constituent, the final feed mixture being prepared atthe size of use, or the yeast cells being administered per se to animalswhich are otherwise fed with normal or adapted feed mixtures.

The yeast cells or yeast cell parts ere suitably and normally mixed withother nutrient components which are preferably selected from protein andcarbohydrate sources, fats or oils and micronutrients such as vitaminsand minerals. As examples of protein sources may be mentioned casein,albumin, wheat gluten, fish meals, concentrated fish residues (fish gluemeal and blood meal). As examples of carbohydrate sources may bementioned gelatinized starch, extruded wheat, molasses, vegetable floursand corn starch. The fat constituents in the feed may for example befish oil and cod liver oil and/or vegetable oils such as corn oil. Theminerals may be selected. e.g., from inorganic or simple organiccompounds of calcium, phosphorus, sodium, potassium, chlorine,magnesium, copper, manganese, zink, cobalt and selenium. As examples ofvitamins may be mentioned vitamin B₁₂, proline, vitamin A, vitamin D,vitamin E, vitamin K, thiamine, ascorbic acid, riboflavine, pyridoxine,panthotenic acid, niacine, biotin, choline and inositol.

The invention also relates to food or feed comprising astaxanthin whichhas been extracted from yeast cells, for example by any of the methodsdescribed above, preferably from yeast cells according to the inventionor yeast cells produced by the method of the invention. The astaxanthinmay be used in admixture with the feed constituents described above andalso in admixture with other food or nutrient components as well as inadmixture with other colourants. Thus, astaxanthin extracted from yeastcells is well suited alone or in combination with other colourants foruse in edible oils, butter, margarine, shortening, mayonnaise, pates,soups, snack products, surimi-based products, desserts, ice cream,confectionery, baked products, and beverages. When the astaxanthin isused in food which is mostly constituted by water or water phases, theastaxanthin is preferably mixed with an emulsifier as discussed abovewhich makes the astaxanthin dispersible in the water phase without anytendency to crystallize and without the necessity of adding an oilyphase to dissolve the astaxanthin.

Furthermore, the invention relates to a method for feeding animals toobtain a reddish pigmentation of their meat and/or of products producedby the animals, comprising administering to the animals a feedcontaining yeast cells or cell parts containing astaxanthin in an amountof at least 300 μg per g of yeast dry matter, determined by the methodstated above, or astaxanthin extracted from such yeast cells or cellparts.

The amount of the feed containing the astaxanthin or theastaxanthin-containing yeast cells or cell parts administered to theanimals will depend upon the animal species in question and upon thepigmentation effect which it is desired to obtain by means of theastaxanthin. Evidently, the principle to be followed is that the animalshould have a normal recommended daily ration of macro- andmicronutrients and, in addition, astaxanthin in a form and an amountwhich will result in the desired pigmentation of the animal meat or theanimal product in question. In some cases, the amount of astaxanthin tobe administered will depend on the season; thus, for example, it willnormally not be preferred to administer astaxanthin or other carotenoidsto cows to obtain a pigmentation of the butter in the summertime as thebutter pigmentation is normally considered adequate when the cows aregrazing. Also the amount in which the feed containing the astaxanthin orthe astaxanthin-containing yeast cells or cell parts is administered tothe animals may in some cases be dependent on the season. Thus, forexample in the case of fish such as salmon or sea trout, the amount offeed consumed by the fish in the wintertime is relatively low which isin contrast to the consumed by the fish in the summertime. However, asuitable amount of feed administered to the fish may be about 1.5% offish body weight per day which corresponds to the recommendations givenby the California State Department of Fish and Game.

When feeding poultry by the method stated above in order to pigment theyolks of the eggs produced by the poultry and/or the meat or skin of thepoultry, the feed may be constituted by conventional poultry feedcomponents, an example of which is one which is preferably constitutedby protein and carbohydrate sources such as soy bean meal, soy beanprotein, cellulose, starch and fat sources such as soy bean oil,vitamins such as an overall vitamin mix and minerals such as a mixtureof the common mineral components for poultry as well as calcium sourcesfor the egg shells, the calcium sources preferably being calciumcarbonate and calcium hydrogen phosphate. A small amount of sodiumchloride may also be present. The feed may be administered in aconventional dosage.

The invention is further illustrated in the following Examples:

MATERIALS AND METHODS

Maintenance of cultures

Cultures of Phaffia rhodozyma are maintained in two ways:

1) On agar slants (YM-agar). The slants are incubated for one week at20° C. and maintained at 4° C. for 1 month, recultivated in YM-broth,before new slants are made.

2) Cryopreservation at -80° C. From cryovials or agar slants the strainsare inoculated in 50 ml of YM-broth in 250 ml shake flask. The shakeflask is incubated on an orbitshaker (150 rpm) at 20° C. for 4-5 days.The yeast cells are allowed to settle and the liquid is decanted. Thesediment is mixed with glycerol to a concentration of 20%, dispensed incryovials and stored in a deep freezer at -80° C.

Determination of yeast dry matter content

5 ml of a yeast cell culture are centrifuged in a weighed out Sarstedttube (which has been .dried to constant weight at 110° C.) at 10,000×gfor 5 minutes and washed twice in demineralized water. The liquid isremoved by decantation and the weight of the tube with the cells ismeasured after drying to constant weight at 110° C., which gives theweight of the yeast cells (Y g). The yeast dry matter content (YDMC) isthen calculated as:

    YDMC (g/l)=Y/5.00×1,000

The content stated being the mean value of two determinations.

Spectrophotometrical analysis for total pigment determination

The total pigment content in a methanol extract is determinedspectrophotometrically by means of λ_(max) and Beer's law as describedby B. H. Davies, "Carotenoids", in T. W. Goodwin (ed.), Chemistry andBiochemistry of plant pigments, New York, 1976, Vol. 2, p. 149. thespectrophotometer employed is a Shimadzu UV visible recordingspectrophotometer UV 260. Pigment content is calculated by usingformulas 1, 1a, 2, 2a below and the extinction coefficients of Table 1below.

                  TABLE 1                                                         ______________________________________                                        Extinction coefficients of the astaxanthin standard in different              solvents prepared as stated for the standard solution above                                    Absorption                                                                              .sub.E 1%                                          Solvent          maximum   1 cm                                               ______________________________________                                        Acetone          475       2105                                               Methanol         472       2100                                               Ethanol          476       2100                                               Glacial acetic acid                                                                            482       1856                                               ______________________________________                                         .sub.E 1%  absorbance of 1% (w/v) solution in a 1 cm cuvette.                 1 cm                                                                     

Pigment extraction and analysis--Method 1

About 30 ml of the yeast culture were transferred to Sarsteds tubes andcentrifuged for 5 minutes at 10,000×g. The yeast cells were washed indemineralized water and suspended in about 20 ml of methanol. To a glassball mill of the type Bead Beater (Biospec Products Inc., USA) in whichthe rotor was covered with glass balls with a diameter of 0.4 mm (about15 g of glass balls), the methanol suspension was added so as to occupythe remaining free ball mill volume. Disintegration treatment wascarried out by running the mill 5 times for 1 minute at intervals of 30seconds, ice water being kept in the cooling jacket so as to ensure thatthe temperature of the disintegration treatment was kept below 20° C.Immediately after the disintegration treatment, a part of the homogenatewas transferred to Sarstedt tubes, and the yeast dry matter wasdetermined as described above, but without centrifugation. A knownamount (b g) of the homogenate was transferred to a 10 ml measuringflask and solvent was added to give 10 ml. Absorbance was measured inthis solution.

The total pigment content in μg per g of yeast dry matter is determinedby: ##EQU1##

E=absorption at λ_(max) in the solvent used in a 1 cm cuvette

E_(lcm) ^(1%) =absorbance of 1% (w/v) solution in a 1 cm cuvette

b=g of extract

D=mg of yeast dry matter/g extract

Pigment extraction and analysis--Method 2

A predetermined amount of the yeast culture (a ml) were transferred toSarsteds tubes and centrifuged for 5 minutes at 10,000×g. The yeastcells were washed in demineralized water and suspended in about 20 ml ofmethanol. To a glass ball mill of the type Bead Beater (Biospec ProductsInc., USA) in which the rotor was covered with glass balls with adiameter of 0.4 mm (about 15 g of glass balls), the methanol suspensionwas added so as to occupy the remaining free ball mill volume.Disintegration treatment was carried out by running the mill 5 times for1 minute at intervals of 30 seconds, ice water being kept in the coolingjacket so as to ensure that the temperature of the disintegrationtreatment was kept below 20° C. Immediately after the disintegrationtreatment, the liquid was transferred to a 50 ml measuring flask. Theglass beads are washed in the mill with 4×8 ml of methanol. Thefractions are collected and mixed and methanol is added to give 50 ml.The methanol extract is filtered before pigment analysis. The yeast drymatter in the culture is determined as described above.

The total pigment content pr. ml sample is determined by: ##EQU2##

X'=μg of pigment/ml of in sample

a=volume of sample in ml

E=absorbtion at λ_(max) of the solvent used in a 1 cm cuvette

E_(lcm) ^(1%) =absorbance of 1% (w/v) solution in a 1 cm cuvette

The total pigment content per g of yeast dry matter is determined by##EQU3##

Y=μg of pigment/g yeast dry matter

YDMC=g yeast dry matter/l culture broth

HPLC analysis for astaxanthin determination--Method 1

HPLC data:

Equipment:

Columns: LKB Ultropac Precolumn, Lichrosorb RP 18 7 μm, 4×30 mm.

LKB Ultropac Column, Lichrosorb RP 18 5 μm, 4×250 mm.

Detector: LKB 2151 variable wavelength monitor.

Integrator: Waters 740 Data Module.

Controller: LKB 2152 HPLC Controller.

Pumps: LKB 2150 HPLC Pumps.

Autosampler: LKB 2157 autosampler with variable loop.

Manual inj.: Rheodyne 20 μl loop.

Solvents: A: 860 ml of acetonitril+100 ml of water+40 ml of formic acid.

B: 960 ml of ethylacetate+40 ml of formic acid.

All solvents were of HPLC quality.

Flow: 1.0 ml/min.

Gradients : 0-100% B 20 minutes, linear gradient.

100-0% B 10 minutes, linear gradient.

Detector: 471 nm.

Temperature: Ambient temperature.

Standard solution: 5 mg of pure astaxanthin (mp 220°-222° C., absorbanceof 1% v/w acetone solution in a 1 cm cuvette 2100) supplied byHoffmann-La Roche (hereinafter referred to as the astaxanthin standard)are weighed out and dissolved in 500 ml of acetone.

For the HPLC analysis, 20 μl of the sample in question is injected intothe HPLC chromatograph.

HPLC analysis for astaxanthin determination--Method 2

HFLC data:

Equipment:

Columns: Supelco precolumn

Supelco LC 18 - DB. particle size 5 μ

Column dimensions 4.6×250

Detector: LKB 2151 variable wavelength monitor

Integrator: Waters 740 Data Module

Controller: LKB 2152 HPLC Controller

Pumps: LKB 2150 HPLC pumps

Autosampler: LKB 2157 autosampler with variable loop

Manual inj.: Rheodyne 20 μl loop

Solvents: A: 400 ml tetrahydrofurane

400 ml methanol

200 ml 0.02M glycinebuffer pH - 2.6

B: 1000 ml tetrahydrofurane

All solvents except the buffer are of HPLC grade. The buffer issterilfiltered through a 0.22 μm filter before use.

Detector: 480 nm

Temperature: Room temperature

Gradients and flow:

    ______________________________________                                                               Time    Flow                                           Solvent  %             min.    ml/min.                                        ______________________________________                                        B         0             0-11   1.0                                            B         0-90         11-21   1.0                                            B        90            21-29   1.5                                            B        90-50         29-31   1.5                                            B        50            31-32   1.0                                            B        50-0          32-35   1.0                                            B         0            35-39   1.0                                            ______________________________________                                    

Standard solution: 5 mg of astaxanthin standard are weighed out anddissolved in 500 ml tetrahydrofurane.

For the HPLC analysis, 20 μl of the sample in question is injected intothe HPLC chromatograph.

Sarstedt tubes

Polypropylene centrifuge tubes provided with a polypropylene stopper ofthe type 55533 supplied by Hounisens Laboratory, Arhus, Denmark.

Chemicals

Chemicals used in a laboratory scale were of analytical grade. Chemicalsused in fermentations were of food grade.

The glucose and saccharose concentrations were analyzed by of use ofkits (Best. No. 139041) from Boehringer Mannheim.

Medium for shake flask cultivations and agar plates

YM(Yeast Morphology) medium supplied by Difco Laboratories Incorporated(Difco Manual: Dehydrated culture media and reagents for microbiology,10th edition, Detroit, 1984).

Cryo vials

Polypropylene tubes with a volume of 2.0 ml of the type 363401 suppliedby Technunc, Roskilde, Denmark.

EXAMPLE 1

Mutagenization

In each case, the mutagenization treatment was carried out so as toobtain a degree of survival of 1-5% of the treated culture. Suitablemutants were selected by visually comparing the intensity of the redcolour of the mutants when plated as single colonies on the agar plates.

UV-irradation

A just turbid four days old culture of ATCC 24261 grown in YM medium at20°-22° C. was diluted in a 0.9% NaCl solution to concentrations of10⁻¹, 10⁻¹.5, 10⁻², 10⁻².5 and 10⁻³, respectively, and 0.3 ml of each ofthese dilutions was plated on agar plates so as to obtain agar platescontaining 100-300 colonies. The plates were then subjected toultraviolet irradiation at 254 nm for 30 seconds at a distance of 20 cmfrom the irradiation source (Vilbert Lourmat VL 30 LC) and then groom at20°-22° C. for 10 days after which the colour of the resulting colonieswas compared.

EMS (Ethyl-Merhane-Sulphonate) treatment

2×5 ml of a four days old culture of ATCC 24261 grown in YM medium on ashake board at a temperature of 20°-22° C. were centrifugated for 15minutes at 1250×g in a MSE Major centrifuge and the pellet was suspendedin 2×15 ml of sterile 0.9% NaCl solution in 200 ml centrifugation tubes.One of the cell suspensions was employed as a control. To the other cellsuspension 1 g of EMS (Serva 28755) was added. After treatment for 30minutes at 20° C., 150 ml of cold sterile 0.9% NaCl solution was added.The yeast cells were washed twice in sterile 0.9% NaCl and suspended in0.9% NaCl. The yeast cell suspension was then diluted and plated on agarplates in the same manner as described above for the UV-treatment. Oneof the isolated mutants was deposited at the CBS (Centraalbureau voorSchimmelcultures) on 6 Apr., 1987 under the accession No. 224-87.

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine

About 25 mg of N-methyl-N'-nitro-N-nitrosoguanidine (Aldrich Chemie BDR)were added to a 10 ml tared graduated cylinder supplied with a glassstopper. Water was supplied so as to obtain a total volume of 10 ml, andthe N-methyl-N'-nitro-N-nitrosoguanidine was dissolved therein byshaking. 10×1 ml of stock solution were obtained from this solution.

2×20 ml of a four days old culture of CBS 224-87 (the mutant obtained bythe above EMS treatment) grown in YM medium on a shake board 20°-22° C.were transferred to Sarstedt tubes and subjected to two rounds ofcentrifugation. The pellet was suspended in 1.5 ml of 0.9% sterile NaClsolution and 1 ml of the stock solution prepared above was added. Afterincubation for 1 hour at 20°-22° C., the yeast cells were washed 5 timesin 10 ml of cold sterile 0.9% NaCl solution, whereby theN-methyl-N'-nitro-N-nitrosoguanidine was removed. The yeast cells werethen diluted and plated on agar plates in the same manner as describedabove for the UV-treatment. One of the isolated mutants was deposited atthe CBS on 6 Apr., 1987 under the accession No. 225-87.

Reisolation

CBS 225-87 has been subjected to isolation as described as follows. Froma freeze-dried vial yeast cells are suspended in YM medium and incubatedfor 5 day at 20°-22° C. The culture is plated on YM plates and incubatedfor 10 days at 20°-22° C. and new colonies are isolated. One of thecolonies is deposited at the CBS on 23 Mar., 1988 under the accessionNo. 215-88.

EXAMPLE 2

Determination of the astaxanthin content of wild-type Phaffia rhodozymastrains and of mutants prepared in Example 1

The yeast cell cultivation and the astaxanthin determination describedin the present Example constitute, on the one hand, the conditions underwhich the yeast cells are grown and on the other hand the conditionsunder which astaxanthin is determined in the applicant's above-mentionedstandard method for determining the inherent astaxanthin-producingcapability of a yeast strain. These are the same standard conditions asare referred to in the claims.

Shake flask cultivation

100 μl of a 4 days old culture of ATCC 24261 grown in YM medium on ashake board at 20°-22° C. were inoculated in 50 ml of YM mediumcontained in a 500 ml shake flask with 2 baffles. The culture wassubjected to growth on a shake board with orbital shaking at 150 rpm for5 days at 20°-22° C. and at an oxygen transfer rate of 30 mmoles/l/hour,whereby a density of the yeast cell culture of 0.6% was obtained.

Pigment analysis

The astaxanthin content in the extract was identified by the followingthree methods.

1. An acetone, methanol and ethanol extract which had all been preparedas described for the methanol extract preparation above was subjected tospectrophotometric scanning and the λ_(max) values stated in Table 1were obtained.

2. To one half of a 10 ml ethanol extract, prepared in the same manneras above, about 50 mg of potassium borohydride were added to reduce theastaxanthin, and the mixture was stirred for 30 minutes. The absorptionsof the extract and of the potassium borohydride-treated sample weremeasured with varying wavelengths on the spectrophotometer. The freeastaxanthin showed a broad peak at 480 nm and the reduced astaxanthinshowed two peaks at 450 and 476 nm, corresponding to the values statedin the literature.

3. the retention time of the astaxanthin-containing extracts in HPLCunder standard conditions as defined above was compared with theretention time of the standard solution defined above, i.e. The peaks ofthe astaxanthin-containing sample of the invention in HPLC understandard conditions were compared with the peaks of the standardsolution in HPLC. The retention times were found to be identical.

The mutant strains of the invention (CBS 224-87 and CBS 225-87) as wellas all known deposited astaxanthin-producing P. rhozodyma strains weregroom and analyzed in the same manner as described above. The totalpigment content and the astaxanthin content of the strains are stated inTable 2 below.

Total pigment analysis were carried out according to method: pigmentextraction and analysis--Method 1. Astaxanthin were analysed accordingto HPLC analysis for astaxanthin determination--Method 1.

                  TABLE 2                                                         ______________________________________                                                            μg of total                                                                          μg of astaxan-                                                   pigment/g thin/g of                                                           of yeast dry                                                                            of yeast dry                                    Strain              matter    matter                                          ______________________________________                                        CBS 5905 = ATCC 24202 = UCD 67-210                                                                332       254                                             CBS 5908 = ATCC 24203 = UCD 67-383                                                                318       252                                             CBS 6938            303       204                                             CBS 6954            <50       <100                                            ATCC 24201 = UCD 67-203                                                                           229       143                                             ATCC 24203 = UCD 67-383                                                                           338       164                                             ATCC 24228 = UCD 68-653C                                                                          254       107                                             ATCC 24229 = UCD 67-202                                                                           287       142                                             ATCC 24230 = UCD 67-385                                                                           247       132                                             ATCC 24261 = UCD 67-484                                                                           449       286                                             CBS 224-87          885       570                                             CBS 225-87          1176      706                                             ______________________________________                                    

The values are the means of 4 independent measurements. It will be notedthat the mutant strains of the invention show a considerably increasedastaxanthin content.

EXAMPLE 3

Fermentation

The fermentations were performed as fed-batch fermentations undercarbohydrate limitation in thoroughly washed and sterilized 4 m³fermenters of the type Bubble Column with a stationary aeration systemconsisting of perforated air pipes. The fermenters were equipped with pHelectrodes, inlets for pH regulating agents and foam-suppressing agents,and alcohol detectors for measuring alcohol in the discharged air. Thejackets of the fermenters were thermostated.

The start wort has the following composition: 20 g/l of molasses, 0.6g/l of diammonium sulphate, 0.8 g/l of diammonium hydrogenphosphate and0.125 g/l of magnesium sulphate which altogether were boiled up in thefermenter for 30 minutes together with a suitable amount of water (30 1in the 100 1 propagation fermenter and 2000 1 in the 4 m³ productionfermenter) before the fermenter in question was inoculated. The mediumwhich was fed to the fermenter in the fed-batch fermentation was takenfrom two different reservoirs, i.e. a chemical reservoir consisting of10 kg of diammonium sulphate, 5.6 kg of diammonium hydrogenphosphate and80 1 of water; and a molasses reservoir consisting of 450 kg of molassesand 1000 1 of water which had been autoclaved. 0.1 mg of desthiobiotinand 1.6 kg of magnesium sulphate were supplied directly to the fermenterbefore the rest of the medium was supplied. All the chemicals were offood grade. The molasses were beet molasses from De DanskeSukkerfabrikker.

The aeration during the fermentation was 8.4 m³ /minute. Contraspum 210(Zschimmer & Schwartz) was employed as the foam-suppressing agent, andsulfuric acid was employed as the pH regulating agent.

Yeast cells of strain ATCC 24261 were propagated by being transferredfrom a slant to a test tube with a diameter of 2 cm containing 5 ml ofYM medium in which the cells were cultured for 4 days on a shake boardunder sufficient aeration at a temperature of 20°-22° C., after whichthe culture was transferred to 2 1 Erlenmeyer flasks containing 1 1 ofYM medium. After incubation for 3 days on a shake board and undersufficient aeration at a temperature of 20°-22° C., 1 1 of the culturewas transferred to a 100 1 fermenter containing 30 1 of start wort. Theculture was subjected to batch growth at 20°-22° C. until a yeast drymatter content of 1 g/l was obtained. Thereafter, the nutrient supplywas started and the fed-batch fermentation was performed at 20°-22° C.After 2 days' growth, 30 1 of the culture were transferred under sterileconditions by means of a peristaltic pump to the 4 m³ fermenter whichcontained 2000 1 of start wort. The culture was subjected to batchgrowth at 20°-22° C. until a yeast dry matter content of 1 g/l in theculture was obtained. Then, the molasses supply was started andcontinued for 38 hours after which the molasses reservoir was depleted.The chemicals were supplied proportionally with the molasses during thefirst 24 hours. The fed-batch fermentation was performed at atemperature of 20°-22° C. The amount of molasses in the molassesreservoir was adjusted so that a yeast dry matter content of not morethan about 4% in the fermented wort would be obtained.

The molasses supply to the fermenter during the fed-batch fermentationwas adjusted with the aim of reaching a specific growth rate of theyeast cells of μ=0.15 hour⁻¹ and was further regulated in accordancewith the ethanol concentration in the wort which should be lower than0.1% by volume. Thus, the ethanol concentration was frequently measured,and when it was found to be too high, the molasses supply rate waslowered until an acceptable ethanol content was again obtained.

The aeration of the fermented wort was continued for 16 hours at 20°-22°C. without any nutrient supply.

The composition of the yeast cells as well as the total pigment contentand the astaxanthin content were measured at time 0, i.e. just beforethe nutrient supply to the 4 m³ fermenter was started, after 38 hourswhen the fermentation and growth had terminated, and after 16 hours'aeration of the fermented wort. The total pigment content and theastaxanthin content were determined as described in Example 2, and thecomposition of the yeast cells was determined by conventionaltechniques. Thus, the total content of nitrogen was determined byKjeldahl analysis, the trehalose content was determined as described inJourn. Am. Chem. Soc. 72, 1950, p. 2059, and phoric acid content wasdetermined as described in Water and Waste-water, American Public HealthAssociation, Inc., p. 199 (1960). Ethanol analysis was performed byBoidin's method for the determination of small amounts of alcohol (cf.Annal. de la brasserie et de la distillerie, 1924-25, p. 177). Theresults are stated in Table 3 below.

Total pigment analysis were carried out according to method 1.Astaxanthin analysis by HPLC were carried out according to method 1.

                                      TABLE 3                                     __________________________________________________________________________    Fed-batch fermentation of ATCC 24261                                          __________________________________________________________________________    Hours      0   18  24  31  38  45  54                                         μg of total pigment/g                                                                 --  181 218 284 415 471 579                                        of yeast dry matter                                                           μg of astaxanthin/g                                                                   --  110 --  --  230 300 350                                        of yeast dry matter                                                           % w/w yeast dry                                                                          0.08                                                                              0.48                                                                              0.95                                                                              2.75                                                                              3.06                                                                              3.15                                                                              3.29                                       matter                                                                        % v/v ethanol                                                                            --  0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                       pH         4.7 4.4 4.0 4.0 4.5 7.8 8.5                                        % w/w N in yeast dry                                                                     --  --  --  --  6.2 6.1 5.7                                        matter                                                                        % w/w P.sub.2 O.sub.5 in yeast                                                           --  --  --  --  2.7 2.6 2.4                                        dry matter                                                                    % w/w trehalose in                                                                       --  --  --  --  2.6 4.4 11.4                                       dry matter                                                                    __________________________________________________________________________

EXAMPLE 4

In a manner similar to the experiment described in Example 3, fed-batchfermentations with strain ATCC 24261 were carried out, the onlydifferences being that the start wort volume was 1000 1, the inoculum inthe 4 m³ fermenter was 6×1 1 of ATCC 24261 which had been propagated asstated above, and the chemical reservoir consisted of 0.5 kg ofdiammonium sulphate, 2.8 kg of diammonium hydrogenphosphate and 80 1 ofwater, and the molasses reservoir consisted of 250 kg of molasses and1000 1 of water. During the fed-batch fermentation, the nutrient was fedto the fermenter for the first 65 hours, after which the nutrient supplywas terminated and the culture was subjected to aeration for 72 hours.

The results are stated in Table 4 below.

                  TABLE 4                                                         ______________________________________                                        Fed-batch fermentation of ATCC 24261                                          ______________________________________                                        Hours             0         65     137                                        μg of total pigment/g of                                                                     149       379    561                                        yeast dry matter                                                              μg of astaxanthin/g of                                                                       89        231    369                                        yeast dry matter                                                              % w/w yeast dry matter                                                                          0.05      3.5    3.75                                       % v/v ethanol     --        0.0    --                                         pH                4.5       7.8    9.1                                        % w/w N in yeast dry matter                                                                     --        4.1    4.5                                        % w/w P.sub.2 O.sub.5 in dry matter                                                             --        1.9    2.2                                        % w/w trehalose in yeast                                                                        2.6       13.2   12.8                                       dry matter                                                                    ______________________________________                                    

EXAMPLE 5

Experiments with the mutant strain CBS 225-87 of the invention werecarried out in the same manner as described in Example 4 with a startwort volume of 1000 1, the inoculum being 6×1 1 of CBS 225-87 which hadbeen propagated by the method described in Example 3 and with a chemicalreservoir consisting of 5 kg of diammonium sulphate, 2.8 kg ofdiammonium hydrogenphosphate and 80 1 of water. No alcohol was formedduring the fed-batch fermentation, and the nutrient was supplied betweenhours 0-57 after which the culture was subjected to aeration withoutnutrient supply. The yeast cell composition at hour 80 was 6.5% nitrogenin yeast dry matter, 2.5% phosphorous pentoxide in yeast dry matter and6.6% trehalose in yeast dry matter. The total pigment, the astaxanthinand the yeast dry matter were determined during the fed-batchfermentation, giving the values stated in Table 5 below:

                  TABLE 5                                                         ______________________________________                                        Fed-batch fermentation of CBS 225-87                                                       Total pigment                                                                           Astaxanthin                                                    Yeast dry  in      in     in    in                                            matter     sample  yeast  sample                                                                              yeast                                 Hours   g/l        μg/ml                                                                              μg/g                                                                              μg/ml                                                                            μg/g                               ______________________________________                                        0       1.4        0.9     640                                                22      11.2       8.1     720                                                33      18.4       12.5    680                                                37      20.0       15.6    780    11.0  550                                   40      20.8       16.5    790    8.8   420                                   57      23.8       24.0    1010   16.2  680                                   61      24.7       24.1    980    17.0  690                                   64      24.3       25.5    1050   17.3  710                                   80      24.6       36.6    1490   23.6  960                                   ______________________________________                                    

The total pigment and astaxanthin content in μg/ml has been calculatedfrom the analyzed yeast dry matter content and μg/g-values of totalpigment and astaxanthin.

EXAMPLE 6

The pigment and astaxanthin content of CBS 215-88 and P. rhodozymamutant strains DBT 406 and DBT 403, the wildtype strains CBS 5905 andATCC 24261 were determined.

Shake flask cultivations

From an agar slope, yeast cells were inoculated in YM-medium andincubated for 2 days at 20°-22° C. 1 ml of the culture was inoculated in50 ml of YM-medium, contained in 250 ml shake flasks with 4 baffles. Theculture was subjected to growth on a shake board wish orbital shaking at150 rpm for 5 days at 20°-22° C.

Quantitative determinations of total pigment were carried out accordingto method 2. Astaxanthin determination were carried out according tomethod 2.

Example of calculations (for CBS 215-88):

The total pigment in the methanol extract was determined byspectrophotometrical analysis. The absorption of the 50 ml extract minusabsorption of methanol was measured to be E=1.189. The volume of thesample was 29 ml. The total pigment content in the yeast extractcalculated by formula (2) as follows:

    X'=1.160×50/2100/29×10.000 μg/ml=9.52 μg/ml

The yeast dry matter was determined to be 6.7 g/l the total pigmentcontent per g of yeast dry matter is determined by formula (2a) asfollows:

    Y=9.52/7.1×1.000 μg/g=1340 μg/g

the concentration of astaxanthin in the methanol was determined by HPLCanalysis to be 6.4 μg/ml corresponding:

    6.2 μg/ml/7.1 g/l=880 μg astaxanthin/g yeast dry matter

All strains were grown and analysed in the same manner as describedabove. The total pigment and astaxanthin content of the strain arestated in Table 6.

                  TABLE 6                                                         ______________________________________                                               Yeast dry matter                                                                          Total pigment                                                                             Astaxanthin                                    Strain   g/l           μg/ml                                                                             μg/g                                                                              μg/ml                                                                           μg/g                             ______________________________________                                        CBS 5905 5.3           1.38   260    0.84 160                                 ATCC 24261                                                                             5.5           2.10   380    1.38 250                                 DBT 406  2.2           5.84   2650   3.4  1540                                DBT 403  1.6           5.04   3150   3.3  2050                                CBS 215-88                                                                             7.1           9.52   1340   6.2  880                                 ______________________________________                                    

EXAMPLE 7

Fed-batch fermentation of the mutant strain CBS 215-88 of the inventionwere carried out using corn steep solid and sucrose as carbohydratesources in the same 4 m³ fermenter as described in Example 3. the startwort consisting of

10 - kg corn steep solid

12 - sucrose

10 - diammonium sulphate

3 - potasium dihydrogen phosphate

1 - magnesium sulphate

0.05 g biotin

300 ml antifoam Contraspum 210

1000 1 water

was sterilized at pH 4.6 by injection of steam at 95° C. for one hourand after cooling to 22° C. 6×1 1 CBS 215-88 propagated as stated abovewas added as inoculum. After 35 hours' of aeration (4.2 m³ /minute)supply of sucrose solution (0.30 g/l) was started. The addition rate was2.3 1/hour. Aeration was incereased to 8.4 m³ /minute and thepH-controller started (set point 4.0). When the sucrose concentration inthe medium was decreased to about 1 g/l after 28 hours, the sucrosesupply was increased to 7.3 1/hour and kept at this rate for 24 hours.Thereafter sucrose supply was terminated and the aeration rate wasdecreased to 4.2 m^(3/) minute and continued for 72 hours. The totalpigment and astaxanthin were determined during the fed-batchfermentation, giving the values stated in Table 7.

                  TABLE 7                                                         ______________________________________                                        Fed-batch fermentation of CBS 215-88                                          ______________________________________                                        Hours of sucrose supply                                                                         28        52                                                Hours of aeration after            6                                          termination of sucrose supply                                                 μg of total pigment/ml                                                                       14.1      37.7   43.4                                       culture medium                                                                μg of astaxanthin/ml                                                                         --        23.4   29.9                                       culture medium                                                                pH                4.0       4.0    4.0                                        % w/w N in yeast dry matter                                                                     --        --     7.69                                       % w/w P.sub.2 O.sub.5 in yeast dry matter                                                       --        --     3.72                                       % w/w trehalose in yeast dry                                                                    --        --     4.4                                        matter                                                                        ______________________________________                                    

The yeast cells were separated from the medium by means ofcentrifugation in a De Laval OA5M centrifuge and washed with watertwice. The yeast was separated from the yeast cream by means offiltration in a FILTROX-filter, type VARIOX 40/40 cm and the filter cakewith 26.3% dry matter was extruded through a 1 mm sieve in a lab fluidbed dryer (GLATT, Haltingen-Binzen Bd.) and dried at 30° C. for 90minutes. The dried yeast (91.6% of dry matter) contained

    1360 μg total pigment/g yeast dry matter

and

    1080 μg astaxanthin/yeast dry matter

EXAMPLE 8

Downstream processing

Yeast cells obtained by the method of Example 3 were isolated from thefermented wort by centrifugation in a De Laval OA5M centrifuge. Thecells were washed twice with water and a yeast cream with 13% of yeastdry matter was obtained. The pH of :he yeast cream was adjusted to 4.0by addition of sulphuric acid, and sodium benzoate was added to aconcentration of 0.2% (w/v) in the yeast cream. During the treatment ofthe isolated yeast cells, these were under a nitrogen cover so as toprevent substantial oxidation of the astaxanthin of the yeast cells, andthe temperature was kept at about 10° C.

The yeast cream was subjected to three passages through a systemconsisting of an APV-Gaulin MC4 homogenizer provided with a cell rupturevalve and a heat exchanger where the yeast cream was subjected todisintegration at a pressure of 700 bar, whereby the temperatureincreased by 10°-15° C. and subsequent cooling in the heat exchanger soas to obtain a temperature of 15° C. The yeast cream was circulated inthe system at a raze of 250 1/h.

The astaxanthin content in the disintegrated cells was determined asfollows: 0.5 ml of homogenized yeast cream was weighed out andtransferred to a Sarstedt tube and shaken with 5 ml of acetone. Thesample was centrifugated, transferred to a 10 ml graduated cylinder, andwashed 3 times with acetone with intervening centrifugations andquantitative transfers. The total pigment content was determined bymethod 1 and astaxanthin content was determined by HPLC-method 1, andthe content was related to the total dry matter content in the samplewhich was determined by the method explained in Materials and Methodsabove. By comparing the extractable astaxanthin content in he yeastcream homogenized according to the present Example with the content inyeast cream determined by the method 1 where the cells were completelydisintegrated, the degree of disintegration in the present Example wasdetermined to be more than 90% of the total cells.

To the cells thus disintegrated, which cells were covered with nitrogen,7% of sodium caseinate was added at a temperature of about 45° C. whilestirring. The yeast cell homogenate in admixture with sodium caseinatewas then subjected to spray drying in a spray tower of the type Anhydroin which the inlet temperature was 180° C. The yeast cell mass wasatomized by use of a spray wheel and the temperature of the air let outof the spraytower was of a temperature of about 90° C. The resultingyeast powder was recovered by use of a cyclone. The water content in theyeast powder was less than 10% by weight.

EXAMPLE 9

Extraction of total pigment with glacial acetic acid

20 g of non-ruptured Phaffia rhodozyma yeast cells (which had beenfiltered and subsequently extruded into a fluid bed wherein they hadheed dried) having a dry matter content of 95% and containing 523 μgastaxanthin/g of yeast dry matter were introduced into a column of alength of 12 cm and an inner diameter of 2.4 cm. The column was equippedwith a jacket wherein water of a temperature of 75° C. was circulated.At the bottom of the column, a small amount of acid-washed sand (a sandfilter) was arranged on a cotton layer. The yeast cells were extractedwith 5×100 ml of glacial acetic acid at a temperature of 75° C., and theamount of astaxantin in each of the extracts as well as in the extractedyeast cell material (including about 100 ml of glacial acetic acidremaining in the column) was determined. The extracted yeast cellmaterial had been evaporated to dryness (resulting in 16.16 g ofmaterial) before the astaxanthin determination was carried out. Theresults are stated in Table 8 below.

                  TABLE 8                                                         ______________________________________                                        Total astaxanthin                                                             content in yeast cell                                                         prior to extraction                                                                         20 g × 523 μg/g                                                                      10460   μg                                   Astaxanthin content in                                                        1. extract    100 ml × 55.7 μg/ml                                                                  5570    μg                                   2. extract    100 ml × 14.6 μg/ml                                                                  1460    μg                                   3. extract    100 ml × 4.4 μg/ml                                                                   440     μg                                   4. extract    100 ml × 3.1 μg/ml                                                                   310     μg                                   Total astaxanthin             7780    μg                                   content of extracts                                                           Astaxanthin content of                                                                      16.16 g × 22.5 μg                                                                    363.6   μg                                   extracted yeast cell                                                          material                                                                      Total astaxanthin             8143.6  μg                                   content released from                                                         yeast cells by the                                                            extraction                                                                    ______________________________________                                    

Thus, 77.9% (8143.6/10460×100%) of the astaxanthin content of the yeastcells was released by the extraction.

EXAMPLE 10

Rupturing of yeast cells by homogenization in a ball mill

Phaffia rhodozyma yeast cells, which had been dried in a fluid bed andwhich was found to contain 336 μg of astaxanthin/g of yeast dry matter,was suspended in soy bean oil in a concentration of 40% (w/w). Thesuspension was pumped to a ball mill (CoBall® - Mill, type MSZ-12)containing zirkonium balls (0.1-1.5 mm) and having a bead-filling of70-75%. The bead of the rotor was 13 m/sec. Samples were taken aftereach run, and the astaxanthin content of the samples was analyzed onHPLC. The temperature in the ball mill was kept at 40°-50° C.

Similarly, a suspension of the above dried yeast cells in 75% water wastreated in the ball mill. In this treatment, the speed of the rotor was15 m/sec.

The results are stated in Table 8, wherein the astaxanthin content isstated as μg/g of yeast dry matter.

                  TABLE 9                                                         ______________________________________                                                   60% soy bean oil                                                                        75% water                                                ______________________________________                                        1st run      221         179                                                  2nd run      308         192                                                  3rd run      313         276                                                  ______________________________________                                    

For comparison, only 23 μg of astaxanthin/g of yeast dry matter wasfound when the dried yeast was treated with soy bean oil or waterwithout the simultaneous homogenization.

By the experiment it is shown that about 3 runs in the ball mill aresufficient to obtain a substantially total rupture of the yeast cell.

EXAMPLE 11

Feeding of fish

Fish feed of varying astaxanthin contents were prepared. The fish feedwas made from the commercial fish feed Ecoline 16 from Dansk Orredfoder,Brande, Denmark, which is a mixture of fish meal, soy meal, fish oil,extruded wheat, lecitin and vitamins in the form of a premix. Variousamounts of astaxanthin were added to this feed. The astaxanthin wasobtained from P. rhodozyma yeast cells which had been groom in the samemanner as described in Example 3 and which had been spray dried. Thespray dried yeast cells were prepared from 28 kg homogenized yeast creamwhich had been mixed with 0.475 kg of sodium caseinate dissolved in 2.7kg water at about 50° C. 0.068 kg GRINDTEK MOR 50 containing 2 g ofascorbyl palmitate and 1 g of tocopherols from soy beans was emulgatedin the sodium caseinate solution. The sodium caseinate solution(containing antioxidants) was mixed with the homogenized yeast cream andthe mixture was spray dried as described in Example 8. the spray driedproduct (92.6% dry matter) contained about 674 μg of astaxanthin/g ofyeast dry matter. The spray dried product was added to the commercialfish feed so as to obtain the varying concentrations of astaxanthin inthe fish feed (feed A-D) which appear from table 10 below.

A fish feed containing synthetic astaxanthin (feed E) was employed as acontrol. The feed E contained synthetic astaxanthin in an amountcorresponding to 40 ppm.

The feeds A-E had the following composition:

                  TABLE 10                                                        ______________________________________                                        Fish feed                                                                                A      B      C        D    E                                      ______________________________________                                        Astaxanthin μg/kg feed                                                                  4.4      12.8   20.4   39.2 --                                   Synthetic astaxanthin                                                                      --       --     --     --   40                                   Dry matter % 91.94    91.49  91.56  91.88                                                                              88.81                                Ash %        9.12     9.00   8.79   8.45 7.03                                 cellulose %  1.46     1.45   1.60   2.05 1.71                                 Protein %    43.62    43.20  43.86  43.81                                                                              43.15                                Fat %        17.59    17.21  18.62  20.00                                                                              21.19                                Phosphor g/kg                                                                              11.58    10.89  11.25  10.80                                                                              9.96                                 Nitrogen-free extract %                                                                    20.15    20.53  18.69  17.57                                                                              15.73                                ______________________________________                                    

About 60 rainbow trouts, each of a weight of about 400 g, were used inthe experiments with each of the fish feed A-E. The fish were kept incages and fed ad libitum. The water was of a temperature in the range of2.5°-14° C., the lower temperatures in the last part of the fish feedingexperiment.

Extraction of astaxanthin from fish

The equipment used is equipment conventionally used in laboratoryexperiments.

A rainbow trout without skin was cut into pieces and 15 g of flesh wereweighed out in a centrifugal tube (100 ml). 15 ml of tetrahydrofuranewere added as extraction agent. The flesh was further divided in anULTRATURAX mixer and subsequently centrifugated. The tetrahydrofuraneextract was transferred to a 50 ml measuring flask. The remanence waswashed with 10 ml of tetrahydrofurane for 2-3 minutes on a sonicationbath and centrifugated, and the tetrahydrofurane phase was transferredto the measuring flask to which additional tetrahydrofurane was added upto 50 ml. 10 ml out of the 50 ml were subjected to evaporation under anitrogen cover at a temperature of 40° C. The evaporation residue wasredissolved in 1 ml of mobile phase and filtered through a 0.45 μmfilter prior to further analysis.

HPLC analysis

Column: LiChrosorb RP-18, 5 μm, 250×4.6 mm

Mobile phase: 40 ml formic acid

60 ml water

384 ml ethylacetate

516 ml acetonitrile

Flow: 10 ml/min.

Injection: 20 μl loop, Rheodyne 7120

Pump: Waters 510

Detector: Waters 481, UV-spectrophotometer, 471 nm

Integrator: Waters 740

Determination of the colour of the fish

The colour of the fish flesh was determined by the L*a*b*-colourdetermination method by use of a Minolta Chroma Meter II. The L*-valuedesignates the light component, the a*-value (ranging from -60 to +60)designates the green/red component (the negative values designating thegreen component and the positive values designating the red component),and the b*-value (ranging from -60 to +60) designates the blue/yellowcomponent of the colour. Only the a*-value is stated in Table 11.

Rainbow trout flesh without skin was homogenized in a blender to obtaina homogeneous mass which was put into a small petri-dish (of a height of1 cm and a diameter of 3.5 cm) so as to occupy the total volume of this.The surface of the mass in the petri-dish was smoothened out and coveredwith a glass plate, and was then ready for analysis.

In the following Table 11, the data of the fish feeding experiment arestated:

                  TABLE 11                                                        ______________________________________                                        μg of asta-              μg of                                          xanthin/g          weight of                                                                              fish                                              of fish            fish in g                                                                              flesh (a*)                                        ______________________________________                                        Analysis of fish fed for 16 days                                              Feed A  0.50           380      1.45                                                  0.60           434      1.12                                                  0.65           252      0.08                                                  0.60           580      0.48                                                  0.65           392      -0.02                                         Feed B  1.45           343      1.53                                                  0.15           385      0.05                                                  0.20           298      -0.52                                                 1.00           388      0.52                                                  0.60           317      -0.33                                         Feed C  0.75           471      0.00                                                  0.85           290      1.20                                                  0.45           328      0.23                                                  0.30           300      -0.38                                                 0.75           359      -0.38                                         Feed D  0.70           258      -0.20                                                 0.45           643      0.72                                                  1.00           369      2.05                                                  0.70           450      0.50                                                  0.30           382      -0.10                                         Feed E  0.25           374      -0.80                                                 0.60           339      0.12                                                  0.80           405      0.98                                                  0.65           579      0.36                                                  1.20           534      1.18                                          Analysis of fish fed for 23 days                                              Feed A  0.65           668      0.76                                                  0.60           571      1.00                                                  0.40           535      0.26                                                  0.50           469      -0.50                                                 0.20           386      0.06                                          Feed B  0.40           348      0.02                                                  0.40           325      0.16                                                  0.75           500      1.32                                                  0.75           355      0.16                                                  0.50           406      0.18                                          Feed C  1.25           356      1.25                                                  1.05           464      1.88                                                  1.05           317      1.98                                                  0.25           301      -1.44                                                 0.50           329      -0.44                                         Feed D  0.50           287      -0.14                                                 0.65           380      1.34                                                  0.45           436      0.62                                                  1.65           449      3.38                                                  1.85           409      3.26                                          Feed E  2.00           487      4.08                                                  0.50           429      0.82                                                  0.35           673      2.53                                                  1.20           441      3.16                                                  1.15           404      0.90                                          Analysis of fish fed for 30 days                                              Feed A  0.80           410      1.12                                                  0.60           448      0.78                                                  0.50           409      1.50                                                  0.55           483      1.40                                                  0.65           352      0.40                                          Feed B  0.75           344      0.35                                                  0.35           410      1.17                                                  0.45           547      0.40                                                  0.35           493      2.20                                                  0.95           228      2.14                                          Feed C  1.10           517      3.30                                                  0.95           405      1.48                                                  1.55           381      2.26                                                  0.75           330      1.48                                                  0.95           413      1.80                                          Feed D  2.15           635      6.10                                                  0.85           384      1.68                                                  2.10           363      0.56                                                  1.70           423      3.70                                                  1.00           348      1.92                                          Feed E  1.15           390      1.84                                                  2.10           427      4.00                                                  3.00           433      4.14                                                  0.40           337      0.02                                                  0.55           342      0.85                                          Analysis of fish fed for 43 days                                              Feed A  1.00           429      2.50                                                  0.60           300      -0.56                                                 0.90           848      2.32                                                  0.50           417      0.16                                                  0.45           385      -0.54                                         Feed B  0.75           623      1.94                                                  1.00           352      1.88                                                  1.00           620      1.44                                                  0.75           484      1.52                                                  0.95           441      0.78                                          Feed C  0.75           604      1.30                                                  0.90           480      1.84                                                  1.20           540      2.96                                                  2.20           444      4.78                                                  1.35           414      0.74                                          Feed D  1.45           471      3.66                                                  2.10           436      6.61                                                  2.15           508      5.56                                                  3.00           510      6.04                                                  1.15           381      1.72                                          Feed E  1.20           512      2.40                                                  3.00           452      4.84                                                  4.30           634      8.04                                                  1.75           474      4.43                                                  3.70           517      5.46                                          Analysis of fish fed for 72 days                                              Feed A  0.55           409      1.08                                                  0.70           628      4.02                                                  1.95           1177     3.84                                                  0.70           663      1.42                                                  1.65           401      1.34                                          Feed B  1.05           386      2.24                                                  0.90           666      2.46                                                  0.30           507      2.46                                                  2.05           585      4.90                                                  1.20           518      1.52                                          Feed C  1.15           701      2.40                                                  1.80           415      4.42                                                  4.80           739      9.36                                                  5.00           451      7.26                                                  4.00           594      5.48                                          Feed D  1.15           444      2.32                                                  2.05           514      6.98                                                  4.25           493      8.40                                                  4.80           612      8.24                                                  4.25           633      6.66                                          Feed E  3.05           627      6.32                                                  3.75           618      6.26                                                  4.65           507      9.78                                                  5.95           654      8.56                                          ______________________________________                                    

The results show that the astaxanthin of each of the fish feed A-E hasbeen absorbed by the fish and that the fish flesh obtains an increasingred pigmentation with increasing amounts of astaxanthin in the feed(feed A-D) and with increasing time of feeding (as observed by thea*-value (designating the red component of the colour)).

Further, the above results indicate that the presence of astaxanthin inthe feed do not affect the growth of the fish.

The fish were also subjected to visual examination and were generallyfound to be of an attractive red colour. After 43 days of feeding,substantially no difference was observed in the pigmentation of fish fedwith feed D and E (containing about 40 ppm astaxanthin producedaccording to the present invention and 40 ppm synthetic astaxanthin,respectively). After 72 days of feeding, substantially no difference wasobserved in the pigmentation of fish fed with feed C, D and E(containing about 20 ppm astaxanthin produced according to the presentinvention, 40 ppm astaxanthin produced according to the presentinvention, and 40 ppm synthetic astaxanthin, respectively).

EXAMPLE 12

Fish pate

A fish pate (salmon-like) in which astaxanthin is used to impart the redcolour can be made according to the following recipe:

    ______________________________________                                        Cod scraps              71%                                                   Oil                     3%                                                    Rusk                    4%                                                    Grindsted Protein 177*.sup.)                                                                          1%                                                    Starch                  1%                                                    Astaxanthin             0.001%                                                Water, preservatives and                                                                              100%                                                  spices up to                                                                  ______________________________________                                         *.sup.) Grindsted Protein 177 is a blend of 75% of Grindsted Protein 100      and 25% of sodium alginate.                                              

The astaxanthin was dispersed in the oil phase. The Grindsted Protein177, starch and other dry ingredients were mixed, and the fish wereadded to a colloid mill. Then, the oil phase, the dry ingredients andwater were added, and processing was continued in the colloid mill forabout 10 minutes. The pate was filled into tins and subjected to heattreatment.

Red dressing

A red dressing with an attractive red colour can be prepared byconventional methods from the following ingredients:

    ______________________________________                                        Oil                    30.0%                                                  Tarragon vinegar       12.3%                                                  Tomato paste           8.0%                                                   Mayodan DC*.sup.)      0.3%                                                   Sugar                  8.0%                                                   Salt                   0.8%                                                   Astaxanthin            0.01-0.5%                                              Water, preservatives and                                                                             100%                                                   spices up to                                                                  ______________________________________                                         *.sup.) Mayodan DC ® is a stabilizer blend from Grindsted Products        A/S.                                                                     

We claim:
 1. An isolated pure culture of a strain of Phaffia rhodozymawhich when grown under conditions comprising an oxygen transfer rate ofat least 30 mmoles/1/hour on YM medium at 20°-22° C. for 5 days in 500ml shake flasks with two baffles containing 50 ml of the medium andsubjected to orbital shaking at 150 rpm, produces astaxanthin in anamount of at least 600 μg per g Phaffia rhodozyma dry matter, asdetermined by HPLC analysis.
 2. The culture according to claim 1 whereinsaid strain produces astaxanthin in an amount of at least 700 μg per gof Phaffia rhodozyma dry matter.